ABSTRACT During T cell interaction with APC, CD28 is recruited to the central region (cSMAC) of the immunological synapse. CD28-mediated signaling through PI3K results in the recruitment of protein kinase C-theta (PKCtheta) to the cSMAC, activation of NF-kappaB, and up-regulation of IL-2 transcription. However, the mechanism that mediates CD28 localization to the cSMAC and the functional consequences of CD28 localization to the cSMAC are not understood. In this report, we show that CD28 recruitment and persistence at the immunological synapse requires TCR signals and CD80 engagement. Addition of mAb to either MHC class II or CD80 results in the rapid displacement of CD28 from the immunological synapse. Ligand binding is not sufficient for CD28 localization to the immunological synapse, as truncation of the cytosolic tail of CD28 disrupts synapse localization without effecting the ability of CD28 to bind CD80. Furthermore, a single point mutation in the CD28 cytosolic tail (tyrosine 188) interferes with the ability of CD28 to preferentially accumulate at the cSMAC. PKCtheta distribution at the immunological synapse mirrors the distribution of tyrosine 188-mutated CD28, indicating that CD28 drives the localization of PKCtheta even when CD28 is not localized to the cSMAC. Mutation of tyrosine 188 also results in diminished activation of NF-kappaB, suggesting that CD28-mediated localization of PKCtheta to the cSMAC is important for efficient signal transduction. These data reinforce the importance of the interplay of signals between TCR and CD28 and suggest that CD28 signaling through PCKtheta may be mediated through localization to the cSMAC region of the immunological synapse.

[Show abstract][Hide abstract]ABSTRACT:
T cell activation takes place in the context of a spatial and kinetic reorganization of cell surface proteins and signaling molecules at the contact site with an antigen presenting cell, termed the immunological synapse. Coordination of the activation, recruitment, and signaling from T cell receptor (TCR) in conjunction with adhesion and costimulatory receptors regulates both the initiation and duration of signaling that is required for T cell activation. The costimulatory receptor, CD28, is an essential signaling molecule that determines the quality and quantity of T cell immune responses. Although the functional consequences of CD28 engagement are well described, the molecular mechanisms that regulate CD28 function are largely unknown. Using a micropipet adhesion frequency assay, we show that TCR signaling enhances the direct binding between CD28 and its ligand, CD80. Although CD28 is expressed as a homodimer, soluble recombinant CD28 can only bind ligand monovalently. Our data suggest that the increase in CD28-CD28 binding is mediated through a change in CD28 valency. Molecular dynamic simulations and in vitro mutagenesis indicate that mutations at the base of the CD28 homodimer interface, distal to the ligand-binding site, can induce a change in the orientation of the dimer that allows for bivalent ligand binding. When expressed in T cells, this mutation allows for high avidity CD28-CD80 interactions without TCR signaling. Molecular dynamic simulations also suggest that wild type CD28 can stably adopt a bivalent conformation. These results support a model whereby inside-out signaling from the TCR can enhance CD28 ligand interactions by inducing a change in the CD28 dimer interface to allow for bivalent ligand binding and ultimately the transduction of CD28 costimulatory signals that are required for T cell activation.

[Show abstract][Hide abstract]ABSTRACT:
The interaction between T cells and APCs bearing cognate antigen results in the formation of an immunological synapse (IS). During this process, many receptors and signaling proteins segregate to regions proximal to the synapse. This protein movement is thought to influence T cell function. However, some proteins are transported away from the IS, which is controlled in part by ERM family proteins. Tim-1 is a transmembrane protein with co-stimulatory functions that is found on many immune cells, including T cells. However, the expression pattern of Tim-1 on T cells upon activation by APCs has not been explored. Interestingly, in this study we demonstrate that the majority of Tim-1 on activated T cells is excluded from the IS. Tim-1 predominantly resides outside of the IS, and structure/function studies indicate that the cytoplasmic tail influences Tim-1 polarization. Specifically, a putative ERM binding motif (KRK 244-246) in the Tim-1 cytoplasmic tail appears necessary for proper Tim-1 localization. Furthermore, mutation of the KRK motif results in enhanced Tim1-mediated early tyrosine phosphorylation downstream of TCR/CD28 stimulation. Paradoxically however, the KRK motif is necessary for Tim-1 induced NFAT/AP-1 activation and co-stimulation of cytokine production. This work reveals unexpected complexity underlying Tim-1 localization and suggests potentially novel mechanisms by which Tim-1 modulates T cell activity.

Data provided are for informational purposes only. Although carefully collected, accuracy cannot be guaranteed.
The impact factor represents a rough estimation of the journal's impact factor and does not reflect the actual
current impact factor.
Publisher conditions are provided by RoMEO. Differing provisions from the publisher's actual policy or licence
agreement may be applicable.

migrate through the pSMAC toward the cSMAC. TCR microclus-ters ultimately accumulate at the cSMAC, where they stop signal-ing, and are targeted for down-regulation. In contrast, CD28 sig-naling has been indirectly associated with cSMAC localization.CD28 has been shown to be recruited to the IS and to accumulateat the cSMAC along with TCR (21, 33–35). CD28 is required forrecruitment of protein kinase C-? (PKC?) to the cSMAC region(21, 34). In CD28-deficient T cells or T cells expressing a mutationin the PI3K interaction site, PKC? is still recruited to the IS, but itis not focused into the cSMAC region. This results in a loss inPKC?-dependent activation of NF-?B and up-regulation of IL-2transcription, suggesting that CD28-dependent activation of PI3Kwithin the IS contributes to CD28 costimulation (21). CD28 en-gagement outside the IS (in trans) can still enhance IL-2 secretion,but PKC? is not recruited to either the TCR or CD28 and IL-2 genetranscription is not up-regulated. Rather, CD28 in trans enhancesIL-2 secretion through the induction of IL-2 mRNA stabilization(36). These observations suggest that CD28 localization at the ISmay be an important component for the transduction of some, butnot all, aspects of CD28-mediated T cell costimulation.Little is known about the molecular mechanisms involved in therecruitment of CD28 to the IS. CD28 distributes around the plasmamembrane of nonpolarized T cells and, within minutes of T cell-APC interaction, CD28 accumulates to the contact area, concen-trating into the cSMAC. It has been shown that ligand interactionwith CD80 or CD86 is important to promote CD28 recruitment tothe IS (33, 35). But the signals that drive CD28 targeting to thecSMAC and the intracellular sorting motifs within CD28 that me-diate this localization are not understood. In this report, we haveaddressed both of these issues. We show that sustained signalingfrom both TCR and CD28 is required to maintain CD28 within theIS. Furthermore, we have identified a single point mutation attyrosine 188 (Y188F) that diminishes CD28 localization to thecSMAC. The mislocalization of Y188F CD28 was mirrored bycorresponding mislocalization of PKC? and resulted in a reductionin NF-?B nuclear translocation. These observations support themodel that CD28 costimulation may be controlled in part by lo-calization of CD28 to the cSMAC region of the IS.Materials and MethodsCells and reagents6132 Pro cell transfectants expressing class II (I-Ad) alone (ProAd) or incombination with CD80 (ProAd-B7), ICAM-1 (ProAd-ICAM), or both(ProAd-ICAM-B7) and purification of CD4-positive T cells from DO11.10TCR-transgenic BALB/c mice have been previously described (21). Allcell lines were maintained in DMEM (Invitrogen Life Technologies) sup-plemented with 10% FCS, 2 mM glutamine, 0.1 mM nonessential aminoacids, and 50 ?M 2-ME. CD4?T cells (either from CD28?/?or CD28-deficient mice) were stimulated with 0.2 ?g/ml OVA peptide (323–339)presented by irradiated syngeneic spleen cells and 20 U/ml human rIL-2.When noted, CD28-deficient T cells were activated and 1 day later trans-duced with retroviruses containing wild-type (WT) or mutated murineCD28 (16) or WT murine CD28 fused to cyan fluorescent protein (CFP)(CD28-CFP) (35). Additional mutations in CD28, where portions of thecytosolic tail were placed by alanine (ala scan) or where the entire cytosolictail was deleted (?CT), were constructed by overlapping PCR, confirmedby DNA sequencing, and subcloned into the MIGR retroviral vector. All ofthe viruses used (but not CD28-CFP) contain GFP expressed from an in-ternal ribosome entry site (IRES) as a marker for transduced cells. Infectionefficiency ranged from 10 to 60%, and cells were sorted to match the level ofCD28 expression before each experiment. Abs against PKC? (C-18) andNF-?B p65 (A) were purchased from Santa Cruz Biotechnology; Abs againstCD28(37.51),CD80(1G10),andMHCclassI(34-2-12)werepurchasedfromBD Pharmingen and fluorescently labeled; species-specific, secondary Abswere obtained from Jackson ImmunoResearch Laboratories. Abs againstCD28 (37.51), CD80 (1G10), and MHC class II (M5/114) were also purifiedfrom hybridoma supernatants by protein A-Sepharose. Soluble CD80 (rmB7-1/hFc) was purchased from R&D Systems.Immunofluorescence microscopyPeptide-pulsed APC (2 ?g/ml OVA peptide) were centrifuged with T cellsfor 20 s at a relative centrifugal force of 2000 ? g. The cell pellet wasincubated for 5 min at 37°C, resuspended in complete medium, and eitherincubated for various times or immediately plated on poly-L-lysine-coatedcoverslips. After plating, conjugates were incubated for 5 min at 37°C toallow cell binding to the poly-L-lysine. Cells were fixed in 3% (w/v) para-formaldehyde, permeabilized in 0.3% (v/v) Triton X-100, and stained. ForNF-?B localization, the incubation time before plating on poly-L-lysine-coated coverslips was increased to 35 min, and nuclei were labeled withHoechst stain after fixation and permeabilization. Samples were analyzedon a Zeiss Axiovert microscope controlled by SlideBook software (Intel-ligent Imaging Innovations). Nearest-Neighbor deconvolution, digital anal-ysis, and three-dimensional rendering were accomplished using SlideBooksoftware. The threshold for the images was set using the APC (CD28 andPKC?) or the T cell (MHC class II) fluorescent intensity as nonspecificstaining since these cells should not express the corresponding proteins.CD28 and PKC? distribution within the IS was calculated on a midplaneimage by measuring the length of the cell-cell contact that contained CD28or PKC? and dividing it by the length of the entire interaction site betweenthe T cell and the APC. NF-?B localization to the nucleus was quantifiedby defining a nuclear mask (Hoechst staining) and calculating the fluores-cence intensity of NF-?B staining within the mask area.Live cell microscopyProAd-ICAM-B7 were plated on Delta T culture dishes (Bioptechs) andincubated overnight at 37°C. Ag (2 ?g/ml) was loaded for 2 h and the dishwas mounted on a heated stage maintained at 37°C. CD28-CFP-expressingT cells were added and allowed to interact with the APC. Conjugates wereselected based on sustained recruitment of CD28 toward the APC. Imageswere collected every 21 s for 5 min, 2 ?g/ml control (anti-MHC class I) orblocking (either anti-CD80 or anti-MHC class II) mAbs were added andimaging was continued for an additional 5–20 min.Calcium signalingCalcium imaging of T cell:APC conjugates was done essentially as previ-ously described (37). ProAd-ICAM-B7 cells were preincubated with 2.0?g/ml OVA peptide for 2 h at 37°C and labeled with Alexa Fluor 633(Molecular Probes). T cells were loaded with 1 ?M Indo-1AM (MolecularProbes) for 30 min at 37°C. T cells and APC were mixed at a 1:1 ratio insolution and run on an LSRII flow cytometer (BD Bioscience) to establisha baseline for intracellular calcium levels in the T cells. The T cell:APCmixture was then centrifuged for 15 s at a relative centrifugal force of2000 ? g at room temperature in a microfuge. The cell pellets were re-suspended in warm medium and run on the flow cytometer at 37°C. Tcell:APC conjugates were identified by dual Indo-1/Alexa Fluor 633 flu-orescence, and intracellular calcium levels were determined by ratiometricanalysis of Indo-1-Blue to Indo-1-Violet fluorescence using FlowJo soft-ware (Tree Star).ResultsCD28-ligand interactions are required to recruit and sustainCD28 at the ISCD28 colocalizes with TCR at the cSMAC of the IS in a ligand-dependent manner (33, 35). The requirement for CD28 engage-ment with CD80 to recruit CD28 to the IS can be shown by in-teraction of T cells with APC that do and do not express CD80.Endogenous CD28, detected by anti-CD28 staining, is only re-cruited to the IS in conjunction with CD80-positive APC (Fig. 1,A and B). Retroviral transduction of CD28-deficient T cells with aCD28-CFP fusion protein also demonstrates efficient recruitmentof CD28 to the IS only with CD80-positive APC (Fig. 1, A and B).Similarly, CD28 recruitment to the IS was disrupted when ligandbinding was blocked by the preincubation of APC with anti-CD80mAb, while no effect was observed by the addition of a controlanti-class I mAb (Fig. 1, C and D).To determine whether continuous engagement with ligand isrequired to sustain CD28 at the IS, we allowed CD28 to be re-cruited at the IS and then added anti-CD80 mAb to block CD80-CD28 interactions (Fig. 2A). Within 5 min of anti-CD80 addition,7640 CD28 LOCALIZATION TO THE IMMUNOLOGICAL SYNAPSE by guest on June 13, 2013http://www.jimmunol.org/Downloaded from

Page 4

the majority of conjugates lack CD28 localization at the IS. Ad-dition of a control anti-class I mAb has no effect on CD28 accu-mulation at the IS. TCR signals remained intact when CD80-CD28interactions were blocked, because this treatment does not inhibitcalcium influx on T cells (data not shown). The rapid displacementof CD28 from the IS after anti-CD80 mAb addition can be visu-alized in real-time using T cells expressing the CD28-CFP chimera(Fig. 2B and video 1 in supplemental data4). CD28-CFP-positive Tcell:APC were imaged for 5 min to confirm that CD28 was stablyrecruited to the IS. Blocking anti-CD80 mAb was added and theconjugates were followed for an additional 15–20 min. Approxi-mately 5 min after mAb addition, CD28-CFP began to diffuseaway from the APC and by 10 min was distributed evenly aroundthe T cell surface. In contrast, CD28-CFP remained polarized to-ward the APC the entire period of time when anti-class I mAbwere added (Fig. 2C and video 2 in supplemental data). Theseresults demonstrated the crucial requirement of CD28-CD80 in-teractions not only to initially recruit, but also to maintain, CD28within the IS.CD28 requires constant TCR signal to persist at the ISTo determine whether TCR engagement is required to sustainCD28 at the IS, TCR-peptide-class II interactions were blockedwith the addition of anti-class II-blocking mAb. It has been shownthat this treatment leads to the rapid inhibition of calcium influx,prevention of new TCR microcluster formation, and cessation ofsustained TCR signaling (32, 38, 39). To confirm the efficacy ofanti-class II blocking, conjugates between Indo-1-loaded CD4?Tcells and Alexa Fluor 633-labeled ProAd-ICAM-B7 were formedand the magnitude of the T cell calcium flux in T cell:APC con-jugates was determined on the flow cytometer (Fig. 3, A–C).Blocking amounts of anti-class II mAb were added and conjugateswere analyzed to simultaneously measure the effect of anti-class IIon intracellular calcium flux and on T cell:APC conjugate stabilityover time. After a lag of several minutes, calcium influx was rap-idly inhibited in the entire population of T cells (Fig. 3, B and C).In contrast, the majority of T cell:APC conjugates were stable forat least 15 min after the inhibition of calcium signaling (Fig. 3D).This provides a kinetic window where we could evaluate the re-quirement for sustained TCR signaling in CD28 localization to the ISwithout disrupting T cell:APC interactions. To determine whetherCD28 remains recruited to the IS when TCR signals were blocked,T cells:ProAd-ICAM-B7 conjugates were formed in the presenceof Ag and incubated for 10 min to allow IS formation and CD28recruitment toward the APC. Blocking Abs for class II were addedand conjugates were fixed 5 or 10 min later (Fig. 3E). As a neg-ative control, conjugates were treated with anti-class I mAb. CD28remained recruited to the APC contact area in most of the conju-gates 5 min after the addition of anti-class II mAb. However, after10 min of TCR blocking, localization of CD28 to the IS was lostin most of the conjugates.To visualize CD28 displacement from the APC when TCR sig-nals are blocked, we performed a live cell-imaging analysis.CD28-CFP-expressing T cells were allowed to interact with Ag-pulsed APC for 10 min. Conjugates were selected for CD28 lo-calization to the IS, blocking class II mAb was added, and CD28localization was followed for another 20 min in the presence of theblocking mAb (Fig. 3F and video 3 in supplemental data). CD28remained polarized toward the APC for ?8 min and then rapidlydiffused away, adopting a nonpolarized distribution. Nevertheless,even after complete loss of CD28 polarization within the IS, Tcells remained in close contact with the APC for at least 20 minafter MHC class II-blocking Ab was added. This indicates thatcessation of TCR signaling results in displacement of CD28, well4The online version of this article contains supplemental material.FIGURE 1.retrovirally transduced CD28KO T cells expressing a CD28-CFP fusion protein (CD28-CFP), and Ag-pulsed APC were analyzed based on their ability torecruit CD28 to the IS. CD80-APC are ProAd-ICAM cells that express MHC class II and ICAM-1, but not CD80 or CD86; while CD80?APC areProAd-ICAM-B7 cells that express MHC class II, ICAM-1, and CD80. CD28?/?T cells were stained with anti-CD28 to label endogenous CD28, whilelocalization of CD28-CFP was determined by CFP fluorescence. Representative images (A) and percentage of conjugates that recruit CD28 to the IS (B;n ? 50–65 conjugates) from one experiment representative of two (CD28-CFP) or three (CD28?/?) are shown. C and D, T cell conjugates with Ag-pulsedProAd-ICAM-B7 APC were formed in the presence of 2 ?g/ml mAb against CD80 (anti-CD80) or MHC class I (anti-MHC I) and analyzed for recruitmentof CD28 to the IS. Representative images (C) and percentage of conjugates that recruit CD28 to the IS (D; n ? 50–65 conjugates) from one experimentrepresentative of three is shown. In all of the images, the T cell is oriented toward the top in each panel. ?, p ? 0.0001 by two-population proportion Z-Studentstatistical test.CD28 recruitment to the IS requires ligand binding. A and B, Conjugates between DO11.10 TCR-transgenic, CD4?T cells (CD28?/?), or7641The Journal of Immunology by guest on June 13, 2013http://www.jimmunol.org/Downloaded from

Page 5

before complete dissolution of the IS. Taken together, these dataindicate that steady-state localization of CD28 at the IS depends oncontinuous CD28 ligand binding and TCR signaling.Ligand binding is not sufficient to recruit CD28 to the ISTo identify the cis-elements in CD28 that mediate localization tothe IS, we first generated a truncation of CD28 that lacks the cy-tosolic tail (?CT) (for schematic, see Fig. 5A). The mutation doesnot effect cell surface expression of CD28. WT and ?CT CD28 areexpressed at equivalent levels at the cells surface following retro-viral transduction into CD28-deficient T cells (Fig. 4A). To deter-mine whether the CD28 cytosolic tail is required for recruitment tothe IS, conjugates between retrovirally transduced T cells express-ing either WT or ?CT CD28 and Ag-pulsed ProAd-ICAM-B7were analyzed by microscopy (Fig. 4, C and D). ?CT CD28-ex-pressing T cells showed a significantly reduced ability to recruitCD28 molecules toward the APC. Furthermore, even in those?CT-expressing T cells where CD28 was polarized toward theAPC, significant CD28 was detected outside of the IS, a morphol-ogy that was not detected with cells expressing WT CD28(Fig. 4D).To confirm that the failure to recruit ?CT CD28 to the IS wasnot secondary to a defect in ligand binding, we tested the ability of?CT CD28 to bind CD80. WT or ?CT CD28-expressing T cellswere labeled with a soluble CD80/Ig fusion protein and analyzedby flow cytometry (Fig. 4A). Equivalent levels of CD28 were de-tected on WT and ?CT-expressing T cells by labeling either withanti-CD28 mAb or with the soluble CD80/Ig fusion protein. Dou-ble staining with CD28 and CD80/Ig was not feasible, because theCD28 mAb used blocks the CD80 interaction site. Therefore, weused the IRES-GFP reporter cassette in the retroviral vector tonormalize for levels of retroviral gene expression and then com-pared the cell surface labeling with anti-CD28 mAb and with theCD80/Ig fusion protein for a given level of GFP expression (Fig.4B). WT and ?CT CD28 bound equivalent levels of CD80/Ig fu-sion protein for any given amount of CD28 expression level. Theseresults confirmed that the failure of ?CT CD28 to localize at the ISwas not due to expression levels or inability to interact with CD80,but was a direct result of the cytosolic tail deletion. Furthermore,CD28 localization to the IS is not mediated passively by binding toligand expressed on the surface of the APC. Taken together thesedata indicate that CD28 ligand binding is necessary, but not suf-ficient, to target CD28 to the IS and additional signals from theTCR and cis-elements in the CD28 cytosolic tail are also required.Mutation of tyrosine at position 188 disrupts the ability ofCD28 to focus at the ISTo identify the specific motif within the cytosolic tail of CD28 thatmediates IS localization, we first analyzed a series of amino acidpoint mutations in suspected protein interaction motifs. We tar-geted the YMNM motif at positions 170–173, that upon tyrosinephosphorylation generates a SH2 binding site that has been shownto recruit PI3K and Grb-2 (8, 9, 40). We also targeted prolines at178 and 187/190, that have been implicated in Itk and Lck SH3domain interactions, respectively (11–14) and two additional ty-rosine residues at 188 and 197 (Fig. 5A). Conjugates betweenCD28-deficient T cells expressing different point mutations andAg-pulsed ProAd-ICAM-B7 were formed and analyzed by micros-copy. Strikingly, only one of these mutations showed a significantdefect in its ability to focus CD28 into the IS, a point mutation ofthe tyrosine at position 188 (Fig. 5B). To test the role of additionalsegments of the cytosolic tail, a series of clustered alanine replace-ments were generated (Fig. 5A). Replacement of aa 179–182 and183–186 with alanines had no effect on CD28 localization to theIS. In contrast, alanine substitutions of residues 187–190 (whichcontains Y188) and of residues 192–194 inhibited CD28 localiza-tion to the IS. These results indicate that residues 187–194, andespecially Y188, are required for CD28 focusing within the IS.To more carefully address the role of the cytosolic tail on CD28localization in the IS, we focused our analyses on the single aminoacid mutation Y188F (Fig. 6). T cells expressing Y188F CD28showed a delay in the kinetics of CD28 accumulation at the IScompared with WT CD28 (Fig. 6B). At 15 min, there was a sig-nificant difference in the ability of WT and Y188F CD28 to berecruited at the IS, but by 45 min both WT and Y188F CD28 wererecruited to the IS with similar frequency. However, even whenY188F was recruited to the IS, it was often incomplete and CD28FIGURE 2.A, WT DO11.10 T cell conjugates with Ag-pulsed ProAd-ICAM-B7 APCwere incubated for 10 min to allow IS formation, 2 ?g/ml mAb againstMHC class I or CD80 was added, and the cells were cultured for 5 or 10min before fixation and microscopic analysis. Percentage of conjugates thatrecruit CD28 to the IS (n ? 60 conjugates) is shown. ?, p ? 0.0001 bytwo-population proportion Z-Student statistical test. B and C, Conjugatesbetween CD28-CFP-expressing T cells and Ag-pulsed ProAd-ICAM-B7APC were selected based on CD28 recruitment at the IS. Live cell imagingwas done for 5 min before addition of 2 ?g/ml anti-CD80 (B) or anti-MHCclass I (C) and continued for 15 min after mAb addition. Representativeimages at various time points are shown; complete movies are shown invideos 1 and 2 in supplemental data. The apparent change in morphologyin the T cell following anti-class I addition reflects the T cell crawling ontothe top of the APC (see video 2 in supplemental data). DIC, Differentialinterference contrast.CD28 ligand binding is required to sustain CD28 at the IS.7642 CD28 LOCALIZATION TO THE IMMUNOLOGICAL SYNAPSE by guest on June 13, 2013http://www.jimmunol.org/Downloaded from